Abstract: The effect of Traditional Chinese Medicine (TCM), Ekki-Youketsu-Fusei-Zai
(EYFZ), on human peripheral blood lymphocytes (PBL) was investigated in vitro.
MTT assay showed that EYFZ could directly stimulate PBL to proliferate without
mitogen. In addition, RT-PCR and ELISA revealed that though EYFZ induced the expression
of interferon gamma (INF-γ) mRNA and the production of INF-γ in culture supernatant,
it did not induce the experssion or the production of interleukin-2 (IL-2) or
interleukin-4 (IL-4). Moreover, we also examined which kind of immunocyte was
involved in the INF-γ production by EYFZ in culture supernatant by inhibition
experiment with monoclonal antibodies. It was shown that the EYFZ induced the
production of INF-γ by T cell subsets of CD4＋and CD8＋cells.
Thus, these results suggest that EYFZ has some significant stimulating effects
on proliferation of PBL and the induction of INF-γ by T cells subsets in human.Keywords: Traditional Chinese Medicine (TCM), Ekki-Youketsu-Fusei-Zai (EYFZ),
peripheral blood lymphocytes (PBL), proliferation, interferon-γ(INF-γ) J Saitama Med School 2001;28:125-130
(Received April 24, 2001)

Introduction

Many investigators have reported the efficacy of Traditional Chinese Medicine
(TCM) through various pharmacological and biological studies, using in vivo
and in vitro systems1-3). Ekki-Youketsu-Fusei-Zai (EYFZ),
one of the TCMs, is a mixture composed of six kinds of crude drugs, which have
been used for enhancing the immunological functions of cancer patients in China.
Our studies have shown that EYFZ has anti-tumor effects such as a life-prolonging
effect, tumor-growth inhibition, increase of body weight, and enhancement of
cytotoxicity of natural killer (NK) cells in tumor-bearing mice4).
Also, our study showed that EYFZ could induce the morphological change, the
expression and the production of interleukin-12 (IL-12) on macrophage-like cell
line, J774.15).
However, the effects of EYFZ on human peripheral blood lymphocytes (PBL) have
not been reported so far in vitro. Therefore, in this study, we first
examined whether or not EYFZ could stimulate PBL to proliferate without mitogen,
express cytokine mRNA such as interferon gamma (IFN-γ), interleukin-2 (IL-2)
or interleukin-4 (IL-4) and produce them in culture supernatant. Then, we performed
an inhibition experiment using both anti-CD4 and anti-CD8 monoclonal antibodies
to determine the involvement of CD4＋or CD8＋ T cells in
cytokine production.

Materials
and Methods

Preparation of EYFZ EYFZ is a mixture composed of six kinds of crude
drugs, as reported before4). The crude drugs were obtained from Tochimoto.,
Ltd., Osaka, and EYFZ was prepared as follows: A mixture of Astragali radix
(10.0 g), Zizyphi fructus (5.0 g), Amomi semen (5.0 g), Angelicae
radix (8.0 g), Cervi parvum cornu (5.0 g) and Rehmanniae radix
(10.0 g) was added to 200 ml water and soaked for 20 min. Then, it was boiled
for 30 min for extraction of effective substances, and the solution was centrifuged
at 3000 rpm for 20 min. Finally, the supernatant was condensed to 43 ml (1 g
crude drug/ml) by heating evaporation, and diluted with distilled water to desired
concentrations.Effect of EYFZ on proliferation of human PBL in vitro Effect of
EYFZ on proliferation of human PBL was examined by MTT assay6,7).
Ten ml of peripheral blood was drawn from healthy adult donors into a small
glass tube with heparin, and diluted 1:2 with PBS. Cell suspensions were layered
on a Ficoll Isopaque (1.077 g/ml, Immuno-Biological Laboratories Ltd.) and centrifuged
at 2000 rpm for 30 min. Mononuclear cells were collected from the interphase
and washed three times with PBS. The peripheral blood mononuclear cells were
suspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS), NaHCO3
(2.0 g/L), HEPES (1.0 g/L), L-glutamine (0.6 g/L), and kanamycin (0.25 mg/L),
and cell concentration was adjusted to 1×106 cells/ml. These cells
were incubated in a 75 cm2 tissue culture flask (IWAKI Glass Co., Ltd.) for
2 hr at 37 ℃ in a 5 % CO2 incubator to allow adherence. Then, non-adherent
lymphocytes were gently collected, and cell concentration was adjusted to 5×105
cells/ml. 100 μl of cell susperention was put into each well of 96 well flat-bottomed
plate (IWAKI) with EYFZ (2000 μg/ml, 400 μg/m, 200 μg/ml), concanavalin A (ConA)
(5 μg/ml) (SIGMA CHEMICAL CO.) or culture medium alone, and incubated for 72
hr at 37 ℃ in a 5 % CO2 incubator. After incubation, the proliferation of PBL
was examined by MTT assay: Each well received 10 μl of MTT (SIGMA) solution
(5 mg/ml) and incubated for 5 hr at 37 ℃ in a 5 % CO2 incubator,
then 0.1 ml of 0.04 mol/L HCl-isopropanol was added and incubated for 24 hr
at 37 ℃ in a 5 % CO2 incubator and an absorbance at 570 nm was analysed
spectrophotometrically.Reverse transcription-polymerase chain reaction (RT-PCR) for IFN-c, interleukin-2
(IL-2) and interleukin-4 (IL-4) mRNA expression Human PBL were cultured
with either EYFZ (400 μg/ml), ConA (5 μg/ml) or culture medium alone in 25 cm2
plastic culture flasks (IWAKI) for 72 hr at 37 ℃ in a 5 % CO2 incubator.
After incubation, cells (1×106 cells) were used for the preparation
of RNA. Total RNA was purified using RNeasy Mini Kit (QIAGEN).
The first strand cDNA synthesis was performed by incubating 0.5 μg of RNA
in a total reaction volume of 20 μl containing 1 mM dNTP mixture, 1 mM oligo-(dT),
1x reaction buffer for AMV reverse transcriptase, and 4 units (U) of AMV reverse
transcriptase XL (Life Science) at 42 ℃ for 4 hr. The sequence of oligo-(dT)
was
5'-GCTCTAGATTTTTTTTTTTTTTTTTTTTTT-3'.
RT-PCR was performed by adding 1U Taq polymerase and 2 μl each of 10 mM oligonucleotide
primers as described below. The condition of RT-PCR was as follows: IFN-γ and
IL-2, 45 sec at 94 ℃, 45 sec at 60 ℃, 90sec at 72 ℃ for 35 cycles; IL-4, 70
sec at 94 ℃, 70 sec at 60 ℃, 115 sec at 72 ℃ for 35 cycles.
The following primers were used for RT-PCR:
IFN-γ sense,
5'-ATGAAATATACAAGTTATATCTTGGCTTT-3';
antisense, 5'-GATGCTCTTCGACCTCGAAACAGCAT-3'.
IL-2 sense, 5'-ATGTACAGGATACAACTCCTGTCTT-3';
antisense, 5'-GTCAGTGTTGAGATGATGCTTTGAC-3'.
IL-4 sense, 5'-ATGGGTCTCACCTCCCAACTGCT-3';
antisense, 5'-CGAACACTTTGAATTTTCTCTCTCAT-3'.Analysis of effect of EYFZ on production of IFN-γ, IL-2 and IL-4 by ELISA
Human PBL were cultured under the same condition as described above for the
analysis of production of IFN-γ, IL-2 and IL-4. After 72 hr incubation, the
culture supernatant was collected and stored at -80 ℃ until use. IFN-γ, IL-2
and IL-4 were measured by ELISA according to the manufacturer's instruction
(OptEIATM Human IFN-γ, IL-2 and IL-4 Set, PHARMINGEN) to analyse the effect
of EYFZ on the cytokine production of human PBL.Analysis of lymphocyte subsets involved in IFN-γ production Human PBL
were prepared in a similar way as described above, and adjusted to a cell concentration
of 5×105 cells/ml. Then, the PBL were finally re-suspended in RPMI-1640
medium with or without anti-human CD4(clone H129.19, Pharmingen, Sandiego)(5
μg/ml), anti-human CD8 (clone 53-6.7,Pharmingen, Sandiego)(5 μg/ml) or both
anti-human CD4 and anti-human CD8. These monoclonal antibodies were used in
an inhibition experiment: For example, the activity of cytotoxic T lymphocytes
was shown to be inhibited or blocked by anti-CD88). In parallel with
this test, we also examined the effect of addition of EYFZ (400 μg/ml) to those
using each monoclonal antibody. In this experiment, the control received the
PBL alone or with EYFZ. The cells in 0.2 ml volume per well were incubated using
96 well flat-bottomed plate (IWAKI) for 72 hr at 37 ℃ in a 5 % CO2
incubator. Following incubation, the culture supernatant was collected and stored
at -80 ℃ until use. Then, IFN-γwas similarly measured by ELISA method as described
before to know the effect of monoclonal antibodies on the IFN-γ production with
or without EYFZ.Statistical analysis The data were statistically analysed based on the
Student's t test, and differences were recognized significant with p value less
than 0.05. Here, results were expressed as mean ±standard deviation (SD).

Results

Effects of EYFZ on proliferation of human PBL in vitro Proliferation
of human PBL was examined by MTT assay. Peripheral blood was drawn from healthy
donors, and separated PBL were cultured with either EYFZ (2000 μg/ml, 400 μg/ml,
200 μg/ml), ConA (5 μg/ml) or culture medium alone in a 96 well flat-bottomed
plate. After 72 hr-incubation, the proliferation of PBL was measured by MTT
assay. All samples were measured in triplicate and the experiment was performed
3 times. As shown in Fig. 1, EYFZ directly stimulated PBL to proliferate without
any mitogen, and proliferative effect was almost the same as that of ConA. (P＜0.01
versus the culture medium alone control)

Effect of EYFZ on expression of IFN-γ, IL-2 and IL-4 mRNA Expression
of IFN-γ, IL-2 and IL-4 mRNA of PBL was examined by RT-PCR. As shown in Fig.
2, after 72 hr-incubation, EYFZ (400 μg/ml) induced expression of IFN-c mRNA
as compared with culture medium alone, but not expression of IL-2 and IL-4.
ConA (5 μg/ml), as a control, induced significant expression of IFN-γ, IL-2
and IL-4 mRNA more than in EYFZ addition or in culture medium alone.

Effect of EYFZ on production of IFN-γ, IL-2 and IL-4 in a culture supernatant
It was examined whether or not EYFZ would affect the IFN-γ, IL-2 and IL-4 production
by PBL. As shown in Fig.3, after 72 hr-incubation, EYFZ (400 μg/ml) promoted
significantly the production of INF-γ in culture supernatant of PBL as compared
with that in the culture supernatant without EYFZ or ConA (P＜0.01), but did
not promote IL-2 or IL-4 production. ConA significantly promoted the production
of IFN-γ, IL-2 and IL-4 higher than in EYFZ addition or in culture medium addition
alone.

Fig. 3. Effect of Ekki-Youketsu-Fusei-Zai (EYFZ) on production
of IFN-γ, IL-2 and IL-4 in culture supernatant. Human PBL were cultured
the same conditions as RT-PCR for IFN-γ, IL-2 and IL-4 mRNA expression.
After 72hr incubation, ELISA analysis was used to determine production of
IFN-γ, IL-2 and IL-4. Values are mean ± standard error of triplicates and
represent one of three indepent experiments. (P＜0.01 by Student's two-tailed
test)

Effect of anti-CD4 and anti-CD8 on IFN-γ production with or without EYFZ
This experiment was performed to identify the lymphocyte subsets associated
with INF-γ production by EYFZ. As shown in Fig. 4, though INF-γ was not found
after 72hr culture in the absence of EYFZ, the addition of EYFZ promoted the
INF-γ production. However, the production of INF-γ was inhibited by anti-CD4
or anti-CD8 monoclonal antibodies as compared to the addition of EYFZ without
antibody. The presence of both antibodies also strongly inhibited the INF-γ
production, although this inhibition was not perfect.

Fig. 4. Effect of anti-CD4 and anti-CD8 on IFN-γ production with
or without EYFZ. The human PBL (5×105 cells/ml) were suspended
in RPMI-1640 medium with or without mouse monoclonal anti-human CD4, anti-human
CD8, or both anti-human CD4 and anti-human CD8. 0.2 ml of cell suspension
was put into each well of 96 well flat-bottomed plate with or without EYFZ
(400 μg/ml), and incubated for 72 hr at 37 ℃ in a 5 % CO2 incubator.
After 72 hr incubation, ELISA analysis was used to determine production
of IFN-c. Each column and vertical bar represents the mean ± standard error
of triplicates.

Discussion

In the present study, we examined the effect of EYFZ on the proliferation
and cytokine production of the human PBL. Results clearly showed that culturing
with EYFZ exerted a statistically significant proliferative effect on PBL.
This proliferative response of PBL was comparable to that of ConA. ConA has
been used as the specific mitogen for T lymphocytes9,10). In our
experiment, culturing with EYFZ plus ConA gave similar proliferative effect
on PBL as EYFZ alone and ConA alone (data not shown). This suggests that both
stimulated T lymphocytes and their effects were maximum.
It has been reported that all the stimuli that induce proliferation of T
cells initiate the synthesis of IL-2 and the formation of IL-2R on the cell
surface11). In our experiment, RT-PCR and ELISA revealed that ConA
induced the expression of IL-2 mRNA and the production of IL-2 in a culture
medium, but EYFZ did not induce the expression or the production of IL-2.
About reasons, we don't know exactly now. Because the EYFZ is a mixture of
six kinds of crude drugs and an effective component of each crude drug was
not clearly demonstrated to induce cytokine production. However, some studies
on some murine T cell clones and human PBL shows the stimulation of cell proliferation
with monoclonal antibody12,13). Therefore, since the proliferative
effect of EYFZ on human PBL is possibly due to the IL-2-indepentent pathway.
We will study this possibility in the future.
In addition, we also examined the effects of EYFZ on both the expression
of IFN-γ, IL-2 and IL-4 mRNA and the production of IFN-γ, IL-2 and IL-4 in
culture supernatant. Results showed that EYFZ induced only the expression
of IFN-γ mRNA and production of IFN-γ in culture supernatant. The expressions
and productions of IL-2 or IL-4 were not observed in our experiment. As has
already been demonstrated, helper T cells are classified into two subsets
(Th1 and Th2) on the basis of the cytokines synthesized by T cells. Namely,
Th1 cells secreting IL-2 and/or INF-γ causes the cellular immunity, whereas
Th2 cells14). As our investigation using anti-CD4 and anti-CD8
clearly showed that CD4＋and CD8＋T cell subsets were
at least associated with the IFN-γ production in the presence of EYFZ. There
is one possibility that EYFZ contains some effective components to induce
only IFN-γ from those T cells. The mechanism of only IFN-γ production by T
cells with EYFZ will be needed to be elucidated in the future.
In conclusion, results of our studies suggest that EYFZ can stimulate the
human PBL to proliferate, and induce the expression and production of IFN-c
by stimulating T cells subsets of CD4＋ and CD8＋ T cells.
The effects of EYFZ on T cells of patients in vivo and in vitro
need to be further pursued.